Our recent discovery that specific enzymes can be detected by near infrared optical imaging in vivo was fortuitous and has avalanched a series of research projects to extend preliminary observations The long term goal of this research is to directly image tumoral protease activity (e.g. lysosomal enzymes such as cathepsins or secreted enzymes such as matrix metalloproteinases) in vivo. Endogenous tumor proteases have been implicated in angiogenesis, local aggression and metastases formation and have received attention as therapeutic targets. Exogenous proteases (e.g. from viruses) are also targets for therapeutic intervention and can potentially be used to image gene expression (see Project 3). In the current project we hypothesize that tumor associated proteases, for example cathepsin D, can be used as unique molecular targets for diagnostic purposes to 1) to study protease activity during tumorigenesis, regression and relapse, 2) facilitate molecular characterization of tumors (e.g. metastatic potential) and 3) improve tumor detection. In prior research we have developed first generation auto-quenched near infrared fluorescent (NIRF) probes that become detectable after protease activation and have shown that this quenching/dequenching strategy represents a unique amplification strategy increasing target/background ratio 10-100 fold (Weissleder et al., Nature Biotech 199:375-378). Cell culture, in vitro and in vivo studies confirmed that the probes had very low fluorescence unless activated by proteases and were detectable in nanomole amounts in vivo with no apparent toxicity at concentrations tested. In subsequent studies we have generated second generation probes with enzyme specificity (e.g. cathepsin D), higher fluorochrome payload and better quenching/dequenching characteristics. Using a cathepsin D negative and cathepsin D positive mouse tumor models we have shown the specificity of this approach. The proposed studies are a logical extension of preliminary feasibility studies and will apply the developed technique to imaging of cathepsin D known to be over-expressed 2-50 fold in breast cancers. Overall, we expect that this novel technique will potentially allow to image specific enzymes in living organism which would have important applications in tumor detection and therapy assessment.